Combination Therapy with EGFR Specific CAR_NK_92 Cells and Cabozantinib against Human Renal Cell Carcinoma

Adaptive therapy using immune effector cells engineered by means of chimeric antigen receptors (CAR) has risen as a hopeful cancer management option. Despite their unprecedented success in haematological malignancies, CAR-modified T cells have shown limited efficacy in solid tumours, as the tumor’s immune-suppressive microenvironment inhibits CAR-modified immune effector cells’ functionality by different pathways, counting checkpoint receptor ligands expression like PD-L1 & recruitment tregs like suppressive immune cells. Receptor of epidermal growth factor (EGFR) could be the target of a ll-generation Chimeric antigen receptor T cellshat was transduced to NK-92 cell. In our research, we examined the antitumor efficacy of EGFR specific NK-92 (CAR-NK-92) cells using a xenograft mice model & in conjunction with tyrosine kinase inhibitor cabozantinib. We discovered that EGFR positive renal carcinoma cells (RCC) 786-0 and ACHN may specifically detect and activate CAR_NK_92 cells. They also displayed particular cytotoxicity against RCC in in vitro & in vivo models. Furthermore, we discovered that cabozantinib improves RCC-specific cytotoxicity by enhancing the expression of EGFR while reducing PD-L1 expression in RCC. Our research shows that CAR_NK_92 cells possess anti cancer therapeutic potential for EGFR-positive tumour cells, and that cabozantinib can boost CAR_NK_92 cell cytotoxicity when treated together.

Profiling the inhibitory receptors LAG-3, TIM-3, and TIGIT in renal cell carcinoma reveals malignancy

A cutting edge therapy for future immuno-oncology is targeting a new series of inhibitory receptors (IRs): LAG-3, TIM-3, and TIGIT. Both immunogenomic analyses and diagnostic platforms to distinguish candidates and predict good responders to these IR-related agents are vital in clinical pathology. By applying an automated single-cell count for immunolabelled LAG-3, TIM-3, and TIGIT, we reveal that individual IR levels with exclusive domination in each tumour can serve as valid biomarkers for profiling human renal cell carcinoma (RCC). We uncover the immunogenomic landscape associated with individual IR levels in human RCC tumours with metastases in various organs and histological subtypes. We then externally validate our results and devise a workflow with optimal biomarker cut-offs for discriminating the LAG-3, TIM-3, and TIGIT tumour profiles. The discrimination of LAG-3, TIM-3, and TIGIT profiles in tumours may have a broad impact on investigations of immunotherapy responses after targeting a new series of IRs.

Identification of Appropriate Housekeeping Genes for Gene Expression Studies in Human Renal Cell Carcinoma Under Hypoxic Conditions

Background: Due to the loss of von Hippel-Lindau tumor suppressor function, clear renal cell carcinoma (ccRCC) deregulates hypoxia pathways. Quantitative PCR is a powerful tool for quantifying differential expression between normal and cancer cells. Reliable gene expression analysis requires the use of genes encoding housekeeping genes. Therefore, in this study, eight reference candidate genes were evaluated to determine their stability in 786-0 cells under normoxic and hypoxic conditions. Methods and Results: Four different tools were used to rank the most stable genes: GeNorm, NormFinder, BestKeeper, and Comparative Ct (ΔCt), and a general ranking was performed using the RankAggreg. According to the four algorithms, the TFRC reference gene was identified as the most stable, and therefore, no agreement was observed for the 2nd and 3rd positions. A general classification was then established using the RankAggreg tool. Finally, the three most suitable reference genes to be used in 786-0 cells under normoxic and hypoxic conditions were TFRC, RPLP0, and SDHA. Conclusions: To our knowledge, this is the first study to evaluate reliable genes that can be used in gene expression analysis in ccRcc under a hypoxic environment.

Molecular and Functional Analysis of Sunitinib-Resistance Induction in Human Renal Cell Carcinoma Cells

Resistance in clear cell renal cell carcinoma (ccRCC) against sunitinib is a multifaceted process encompassing numerous molecular aberrations. This induces clinical complications, reducing the treatment success. Understanding these aberrations helps us to select an adapted treatment strategy that surpasses resistance mechanisms, reverting the treatment insensitivity. In this regard, we investigated the dominant mechanisms of resistance to sunitinib and validated an optimized multidrug combination to overcome this resistance. Human ccRCC cells were exposed to single or chronic treatment with sunitinib to obtain three resistant clones. Upon manifestation of sunitinib resistance, morphometric changes in the cells were observed. At the molecular level, the production of cell membrane and extracellular matrix components, chemotaxis, and cell cycle progression were dysregulated. Molecules enforcing the cell cycle progression, i.e., cyclin A, B1, and E, were upregulated. Mass spectrometry analysis revealed the intra- and extracellular presence of N-desethyl sunitinib, the active metabolite. Lysosomal sequestration of sunitinib was confirmed. After treatment with a synergistic optimized drug combination, the cell metabolic activity in Caki-1-sunitinib-resistant cells and 3D heterotypic co-cultures was reduced by >80%, remaining inactive in non-cancerous cells. These results demonstrate geno- and phenotypic changes in response to sunitinib treatment upon resistance induction. Mimicking resistance in the laboratory served as a platform to study drug responses.

The prognostic value of galactosylceramide-sulfotransferase (Gal3ST1) in human renal cell carcinoma

Renal cell carcinoma (RCC) is the deadliest primary genitourinary malignancy typically associated with asymptomatic initial presentation and poorly predictable survival. Next to established risk factors, tumor microenvironment may alter metastatic capacity and immune landscape. Due to their high concentrations, sulfoglycolipids (sulfatides) were among the first well-described antigens in RCC that are associated with worse prognosis. As sulfatide detection in routine diagnostics is not possible, we aimed to test the prognostic value of its protein counterpart, sulfatide-producing enzyme Gal3ST1. We performed retrospective long-term follow up analysis of Gal3ST1 expression as prognostic risk factor in a representative RCC patient cohort. We observed differentially regulated Gal3ST1 expression in all RCC types, being significantly more associated with clear cell RCC than to chromophobe RCC (p = 0.001). Surprisingly, in contrast to published observations from in vitro models, we could not confirm an association between Gal3ST1 expression and a malignant clinical behaviour of the RCC. In our cohort, Gal3ST1 did not significantly influence progression-free survival (Hazard Ratio (HR): 1.7 95% CI (0.6–4.9), p = 0.327). Particularly after adjusting for histology, T-stage, N-status and M-status at baseline, we observed no independent prognostic effect (HR = 1.0 95% CI (0.3–3.3), p = 0.96). The analysis of Gal3ST1 mRNA expression in a TCGA dataset supported the results of our cohort. Thus, Gal3ST1 might help to differentiate between chromophobe RCC and other frequent RCC entities but—despite previously published data from cell culture models—does not qualify as a prognostic marker for RCC. Further investigation of regulatory mechanisms of sulfatide metabolism in human RCC microenvironment is necessary to understand the role of this quantitatively prominent glycosphingolipid in RCC progression.

SPOP Could Play a Potential Inhibitory Role in Human Renal Cell Carcinoma

Background: SPOP, a substrate adaptor of Cul3 ubiquitin ligase, plays crucial roles in solid neoplasms by promoting the ubiquitination and degradation of substrates. Limited studies have shown that SPOP is overexpressed in human renal cell carcinoma (RCC) tissue. However, the exact role of SPOP in RCC remains unclear and needs to be further elucidated. The present study showed that SPOP was expressed at different levels in different RCC cell lines. The purpose of this study was to explore the roles of SPOP in the biological features of RCC cells and determine the expression levels of SPOP in human tissue microarrays (TMAs) and kidney tissues.Methods: Here, SPOP was overexpressed by lentiviral vector transfection in ACHN and Caki-1 cells, and SPOP was knocked down in Caki-2 cells with similar transfection methods. The transfection efficiency was evaluated by quantitative PCR and western blotting analyses. The role of SPOP in the proliferation, migration, invasion and apoptosis of cell lines was determined by the MTT, wound-healing, Transwell and flow cytometry assays. Moreover, the cells were treated with different drug concentrations in proliferation and apoptosis assays to investigate the effect of sunitinib and IFN-α2b on the proliferation and apoptosis of SPOP-overexpressing cells and SPOP-knockdown RCC cells. Finally, immunohistochemical staining of SPOP was performed in kidney tissues and TMAs, which included RCC tissues and corresponding adjacent normal tissues.Results: Overexpression of SPOP inhibited cell proliferation, migration and invasion and increased cell apoptosis. Interestingly, sunitinib and IFN-α2b at several concentrations increased the proliferation inhibitory rate and total apoptosis rate of cells overexpressing SPOP. The findings of the present study showed that the SPOP protein was significantly expressed at low levels in most clear cell RCC (ccRCC) tissues and at relatively high levels in the majority of adjacent normal tissues and kidney tissues. Kaplan-Meier survival analysis showed that there was no statistically significant difference in cumulative survival based on the data of different SPOP expression levels in TMA and patients.Conclusions: In contrast to previous studies, our findings demonstrated that overexpression of SPOP might suppress the progression of RCC cells, which was supported by cell experiments and immunohistochemical staining. SPOP could be a potential tumour inhibitor in RCC.

Overexpression of Capns1 Predicts Poor Prognosis and Correlates with Tumor Progression in Renal Cell Carcinoma

<b><i>Introduction:</i></b> Calpain small subunit 1 (Capns1) has shown its correlation with the metastasis and invasion of hepatocellular carcinoma and intrahepatic cholangiocarcinoma. However, the expression and function of Capns1 in human renal cell carcinoma (RCC) have not been clarified. This study aimed to examine the expression of Capns1 in RCC tissues and cell lines and to assess its role performed in RCC. <b><i>Methods:</i></b> Capns1 expression was evaluated in 75 pairs of RCC and matched adjacent non-tumor tissues by immunohistochemistry. The prognostic value of Capns1 in RCC was assessed by Kaplan-Meier and Cox regression analyses. The action of Capns1 in the proliferation, adhesion, migration, and invasion of RCC cells and the effects on matrix metalloproteinase (MMP) 2 and 9 expression were evaluated after Capns1 silence. <b><i>Results:</i></b> Capns1 expression was significantly higher in RCC tissues compared with the adjacent non-tumor tissues. Multivariate analysis showed that Capns1 overexpression was an independent poor prognostic marker in RCC. The silencing of Capns1 prohibited cell adhesion and impaired the migration and invasion ability of 786-O cells in vitro. Furthermore, Capns1 silence reduced MMP2 and MMP9 expression. <b><i>Conclusion:</i></b> Capns1 overexpression predicts poor prognosis and correlates with tumor progression in RCC. Capns1 expression might serve a prognostic marker and therapeutic target for RCC.